An attempt was made to apply chromatographic techniques, particularly affinity column and high pressure liquid chromatography, to the identification and quantification of substance P in rat brain tissue. The affinity column was made from monoclonal antibody (Ref. 1) linked to CN sepharose 4B. The affinity column was compared to Sep Pak C18 and Silica Sep Pak and found to be superior to both in separating substance P-like immunoreactive peptides from other peptides contained in substantra nigra and hypothalamus of rat brain. An added feature is that the mobile phase of acetomibile/water/phosphoric acid/trithylamine in ratio 28% of: 72%: 100.1% to 0.025% (v/v) used in the HPLC is also excellent for eluting substance P-like peptides that are bound to the antibody in the affinity column. The HPLC method described has a sensitivity of 5-10 mug.